Gene therapy and gene vaccination are techniques that offer interesting possibilities for the treatment and/or prophylaxis of a variety of conditions, as does anti-sense therapy. Such techniques require the introduction of a nucleic acid of interest into target cells. The ability to transfer sufficient nucleic acid to specific target cells remains one of the main limitations to the development of gene therapy, anti-sense therapy and gene vaccination. Both viral and non-viral nucleic acid delivery systems have been proposed. The nucleic acid is generally DNA, but in some cases RNA is used.
The term “gene” is used somewhat loosely in the context of gene vaccination and, especially, gene therapy. While, initially, the term “gene” in those contexts was used to denote the coding sequence of a protein, the term is now used in a general sense to refer to a useful nucleic acid. Examples of nucleic acids that can be used in gene therapy and/or in gene vaccination include the coding sequence of a protein and the cDNA copy and genomic version thereof, the latter including introns as well as exons, and also the regulatory upstream and downstream sequences. Other useful nucleic acids include sequences involved in repairing genes and in homologous recombination. These can be molecules such as RNA/DNA chimeras (Bandyopadhyay et al., 1999; Cole-Strauss et al., 1996; Kren et al., 1998; Yoon et al., 1996) or DNA oligonucleotides (Goncz et al., 1998). A useful nucleic acid can be a short sequence contained in a plasmid, or another large nucleic acid encoding an enzyme that mediates integration of plasmids or nucleic acids, for example, the φC31 phage attB/integrase system (Groth et al., 2000; Olivares et al., 2001; Stoll et al., 2002; Thyagarajan et al., 2000; Thyagarajan et al., 2001) and the “Sleeping Beauty” transposon/transposase system (Yant et al., 2000).
DNA oligonucleotides can be delivered for purposes of antisense regulation (Bachmann et al., 1998; Knudsen and Nielsen, 1997; Mannion et al., 1998; Woolf et al., 1995) or as transcription factor decoys (Ehsan et al., 2001; Ehsan et al., 2002; Mann et al., 1999; Morishita et al., 1995). CpG-rich oligonucleotide sequences may be useful as adjuvants to boost vaccine responses (Krieg et al., 1995).
Another important new class of nucleic acids that can be used in gene therapy includes double-stranded RNA 20-30 nt in length known as small interfering RNA molecules (siRNA). RNA interference in mammalian cells has emerged in the last two or three years as an important new approach to the regulation of gene expression, with a high degree of specificity (reviewed Shi 2003). siRNA molecules target homologous regions of mRNA then activate a conserved pathway that leads to degradation of the mRNA target. The precise mechanism of action of siRNA is under intense investigation but it is clear that the application of siRNA to mammalian cells has the potential to revolutionize the field of functional genomics. The ability to simply, effectively, and specifically down-regulate the expression of genes in mammalian cells holds enormous scientific, commercial, and therapeutic potential.
Currently there is no way to predict an effective siRNA target so screening of numerous sequences is performed and numerous potential molecules may have to be screened. Such screening is most conveniently performed with chemically synthesised siRNA molecules delivered by non-viral vectors. Improved vectors for siRNA transfection would thus provide benefits of cost-effectiveness as well as greater functionality. In vivo use of siRNA molecules in animal models is at a much earlier stage of development but there, too, the potential is enormous.
There are two main modes of transfer of nucleic acid into cells, namely, transfer of naked nucleic acid, and vector-mediated transfer. Non-viral or synthetic vectors fall into three main groups, lipid vectors (lipoplex vectors), vectors comprising other non-lipidic cationic polymers including peptides, dendrimers, and polyethylenimine (PEI) (polyplex vectors), and vectors comprising both cationic polymers and lipids (lipopolyplex vectors) (Felgner et al., 1997). Targeted vectors include viral vectors and receptor-targeted synthetic vectors.
Viral vectors commonly used for gene transfer and hence gene therapy and gene vaccination include genetically engineered, replication-defective derivatives of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus (HSV). They generally exhibit high efficiency of gene transfer in vitro and in some cases, in vivo, in cell types for the which the virus is trophic, i.e., which contain the native receptors. However, gene transfer is poor in cell types that do not contain a native receptor for the virus. Additionally retroviruses are restricted to transducing cells that are dividing rapidly. Furthermore, most viral vectors are restricted in their packaging capacity for nucleic acids, for example, AAV 5 kb; adenovirus 7-8 kb; 35 kb for helper-dependent adenovirus; and retrovirus 10 kb. HSV can package much larger constructs, up to 135-kb (Wade-Martins et al., 2003). Methods of production of replication deficient viral vectors are generally prolonged procedures and in some cases yields of virus are low.
Receptor-mediated gene delivery is a non-viral method of gene transfer that exploits the physiological cellular process of receptor-mediated endocytosis to internalise the nucleic acid. Examples include vectors targeted against insulin receptors, see for example, Rosenkranz et al Experimental Cell Research 199, 323-329 (1992), asialoglycoprotein receptors, see for example, Wu & Wu, Journal of Biological Chemistry 262, 4429-4432 (1987), Chowdhury et al Journal of Biological Chemistry 268, 11265-11271 (1993), and transferrin receptors, see for example, Ciriel et al, Proc. Natl. Acad. Sci. USA 88, 8850-8854 (1991). Further examples of vectors include monoclonal antibodies that target receptors on neuroblastoma cells (Yano et al, 2000), folate conjugated to liposomes (Reddy & Low 2000, Reddy et al. 1999), galactose for targeting liver cells (Han et al. 1999 Bettinger et al. 1999) and asialogylcoprotein, also for liver cells (Wu et al. 1991).
Receptor-mediated non-viral vectors have several advantages over viral vectors. In particular, they lack pathogenicity; they allow targeted gene delivery to specific cell types and they are not restricted in the size of nucleic acid molecules that can be packaged. Gene expression is achieved only if the nucleic acid component of the transfection complex is released intact from the endosome to the cytoplasm and then crosses the nuclear membrane to access the nuclear transcription machinery. However, transfection efficiency is generally poor relative to viral vectors owing to endosomal degradation of the nucleic acid component, failure of the nucleic acid to enter the nucleus and the exclusion of aggregates larger than about 150 nm from clathrin coated vesicles.
Desirable properties of targeting ligands for vectors are that they should bind to cell-surface receptors with high affinity and specificity and mediate efficient vector internalisation. Short peptides have particular advantages as targeting ligands since they are straightforward to synthesise in high purity and, importantly for in vivo use, they have low immunogenic potential.
WO 98/54347 discloses a mixture comprising an integrin-binding component, a polycationic nucleic acid-binding component, and a lipid component, and also discloses a transfection complex comprising
(i) a nucleic acid, especially a nucleic acid encoding a sequence of interest,
(ii) an integrin-binding component,
(iii) a polycationic nucleic acid-binding component, and
(iv) a lipid component.
The transfection complex is primarily an integrin-mediated transfection vector.
It is considered that the components described in WO 98/54347 associate electrostatically to form the vector complex, the vector being of the lipopolyplex type. The vector complexes of WO 98/54347 are found to transfect a range of cell lines and primary cell cultures with high efficiency, with integrin specificity and with low toxicity. For example, vascular smooth muscle cells are transfected with 50% efficiency, endothelial cells with 30% efficiency and haematopoietic cells with 10% efficiency. Furthermore, in vivo transfection of bronchial epithelium of rat lung and pig lung with an efficiency comparable with that of an adenoviral vector has been demonstrated.
Vectors that utilise integrin receptors to mediate gene transfer have the advantage that they target a large number of different types of cells in the body as integrin receptors are relatively widespread. In some circumstances, for example, in in vivo treatment, however, it may be preferable to target recipient cells more specifically.
The dendritic cell is the most potent antigen presenting cell of the immune system and is the only antigen presenting cell capable of stimulating naïve T cell clones, which requires not only recognition of antigenic peptide presented by MHC but also binding costimulatory molecules. The main function of immature dendritic cells is antigen uptake from the surrounding environment. Maturation occurs upon exposure of the cell to danger signals and the function of the cell changes from antigen uptake to peptide presentation on the MHC molecules, combined with trafficking of the dendritic cell to the lymph nodes. Full maturation occurs when the dendritic cells are within the lymph nodes and it is thought that injection or other administration of mature dendritic cells may lead to impairment of homing of the cells.
Transduction or transfection of immature dendritic cells also allows for the introduction of cytokine genes to increase the immune response, whilst also allowing for presentation of peptides taken up from the environment where they have been injected.
Transduction efficiencies to immature dendritic cells using nonviral vectors have been poor, partly due to toxicity. Transfection efficiencies to immature dendritic cells using adenovirus have required high titres of virus, due at least in part to the paucity of the primary adenoviral receptor, the Coxsackie-Adenovirus Receptor (CAR) on the immature dendritic cell surface. Using nonviral vectors, efficiencies have been increased by altering the lipid used. Various strategies have been attempted to increase adenoviral transduction of dendritic cells, including targeting using bispecific antibody fragments (scFv) (Brandao 2003). The use of less adenovirus and a shorter transduction time would be preferable for ex vivo transduction for clinical purposes.
It is an object of the present invention to provide improved vector complexes with enhanced cell targeting properties. The present invention is based on the development of synthetic, targeting non-viral vector complexes that carry a ligand that is more cell-type selective than the ligands of the prior art.
In the development of effective targeting vectors it is useful for several different target-binding ligands to be available. Effective targeted transfection requires not only good targeting but also effective transfer of the vector nucleic acid to the nucleus of the target cell. Even if a ligand is effective in targeting and binding to a target cell, effective gene transfection does not always occur. The reasons for that are, at present, not clear. Accordingly, there remains a degree of unpredictability regarding whether a ligand that binds effectively to a target cell will also bring about effective transfection. It is therefore desirable to have available a “pool” of ligands for any particular cell surface receptor from which an effective transfection ligand may be selected. Such selection may take place by means of a gene transfer assay using, for example, a reporter gene, or by any other suitable means.